Christopher A. Francis

Organic compounds such as urea and cyanate can serve as nitrogen (N) sources for nitrifying microorganisms, including ammonia-oxidizing archaea (AOA) and bacteria (AOB), complete ammonia-oxidizing (comammox) bacteria, and nitrite-oxidizing bacteria (NOB). Here we investigated metagenome-assembled genomes (MAGs) for all four nitrifier guilds generated from hydrologically variable floodplain sediments of the Wind River Basin (WRB; Riverton, WY, USA) for their genetic potential to utilize organic N compounds. A vast majority of WRB nitrifier MAGs harbored urease ( ure ) and at least one urea transporter ( utp , urt , dur3 ). AOA were the most abundant and phylogenetically diverse nitrifiers in WRB floodplain sediments. Several AOA MAGs encoded cyanase ( cynS ), nitrilase ( nit1 ), omega-amidase ( nit2 ), nitrile hydratase ( nthA ), and genes related to purine degradation, including biuret hydrolase ( biuH ), oxamic transcarbamylase ( allFGH ), and catabolic carbamate kinase ( allK ). AOA often encoded an uncharacterized amidohydrolase collocated with biuH , rather than allophanate hydrolase ( atzF ). A small number of AOA encoded atzF , functioning in an unknown pathway. AOB and comammox were of relatively low abundance and taxonomic diversity and were present only at certain depths in WRB; however, they encoded triuret/biuret degradation genes ( trtA , biuH , and atzH ), and in comammox, these genes were also collocated with allFGHK . The genetic potential of ammonia oxidizers in the WRB floodplain suggests that organic N may support nitrification in this system. The proposed pathways for utilizing purine degradation products other than urea potentially expand the known metabolic capabilities of AOA, AOB, and comammox bacteria and reveal the possibility for cryptic N cycling between microbial community members. IMPORTANCE: Floodplains are critical ecosystems where terrestrial and riverine systems meet. Floodplain sediments experience many, sometimes dramatic, changes in moisture and oxygen concentrations because of changes in water table height, flooding, and drought, leading to active microbial cycling of contaminants and nutrients. Nitrogen is one such nutrient that is not only essential for the building blocks of life but can also be used as an energy source by some microorganisms. Microorganisms that oxidize ammonia and nitrite are a crucial part of the nitrogen cycle and can lead to eventual nitrogen loss from a system. Investigating the genes present in microorganisms responsible for nitrification in a dynamic floodplain suggests that organic nitrogen—from decaying plants or potentially other sources, such as fertilizers, grazing livestock feces, or contaminants (e.g., pesticides, pharmaceuticals)—is an important nitrogen source to these microorganisms. This study identifies genes not previously described in nitrifying microorganisms, expanding their potential metabolic substrates.

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle in thaumarchaeota contributes significantly to global organic carbon fixation as the most energetically efficient aerobic carbon fixation pathway. The thaumarchaeal 3-Hydroxypropionyl-CoA Synthetase (ADP-forming; Nmar_1309) is crucial to this efficiency, utilizing ATP to ADP catalysis. This first reported structure of Nmar_1309 reveals a homodimer with a unique subdomain organization ([3-4-1-2-5]) and a distinct linker between subdomains 4 and 1. The presence of bound substrates including 3-hydroxypropionate, non-hydrolyzable ATP (ADPNP), and a phosphate suggests an intermediate state mimicking a reaction step immediately preceding the formation of a 3-hydroxypropionyl-phosphohistidine. Conformational differences were observed between the two chains of the homodimer, likely influenced by the binding of a single ADPNP molecule in one chain. Phylogenetic analysis suggests that while 4HB synthetases may have evolved earlier in the evolutionary timeline, 3HP synthetases in Thaumarchaeota may have occurred after the Great Oxygenation Event. These structural data provide further characterization of the 3HP/4HB cycle and, in conjunction with the structure of 4-hydroxybutyryl-CoA synthetase, Nmar_0206, provide baseline structures of the key ADP-forming Acyl-CoA synthetases within this pathway.

Riparian floodplains are important regions for biogeochemical cycling, including nitrogen. Here, we present MAGs from nitrifying microorganisms, including ammonia‐oxidising archaea (AOA) and comammox bacteria from Slate River (SR) floodplain sediments (Crested Butte, CO, US). Additionally, we explore MAGs from potential nitrite‐oxidising bacteria (NOB) from the Nitrospirales . AOA diversity in SR is lower than observed in other western US floodplain sediments and Nitrosotalea ‐like lineages such as the genus TA‐20 are the dominant AOA. No ammonia‐oxidising bacteria (AOB) MAGs were recovered. Microorganisms from the Palsa‐1315 genus (clade B comammox) are the most abundant ammonia‐oxidizers in SR floodplain sediments. Established NOB are conspicuously absent; however, we recovered MAGs from uncultured lineages of the NS‐4 family ( Nitrospirales ) and Nitrospiraceae that we propose as putative NOB. Nitrite oxidation may be carried out by organisms sister to established Nitrospira NOB lineages based on the genomic content of uncultured Nitrospirales clades. Nitrifier MAGs recovered from SR floodplain sediments harbour genes for using alternative sources of ammonia, such as urea, cyanate, biuret, triuret and nitriles. The SR floodplain therefore appears to be a low ammonia flux environment that selects for oligotrophic nitrifiers.

Estuaries are highly productive ecosystems where microbial communities drive nutrient and carbon cycling, supporting complex food webs. With intensifying anthropogenic pressures, it is critical to understand the capacity of these communities to maintain essential functions under environmental change. Here, we examined the metabolic functions and redundancy in the microbial community of San Francisco Bay (SFB) sediments, providing the first large-scale, genome-resolved, and spatiotemporally resolved characterization of the estuary. Salinity, iron, phosphorus, sulfur, and total sediment nitrogen were significantly correlated with microbial community composition, suggesting these factors play a key role in structuring SFB communities. In support of this, we identified broad capabilities for iron cycling and key uncultured players that contribute to denitrification, nitrification, and complete nitrification (comammox). We also identified widespread capabilities for sulfur cycling, including understudied lineages capable of rDsr-mediated sulfur oxidation. SFB MAGs exhibited partitioning of multistep metabolisms, or metabolic handoffs, and the rare biosphere broadly encoded key nitrogen and sulfur cycling genes. Despite shifts in community composition across sites and fluctuations in environmental parameters, key nitrogen and sulfur metabolisms were maintained throughout the estuary, especially in nitrate reduction, nitrite reduction, and the Dsr/Sox pathway. The presence of multiple microbial taxa with similar functional roles (functional redundancy) may provide an ecosystem buffer, suggesting these functions could better recover from disturbances and ultimately contribute to the long-term health and sustainability of these vital coastal habitats.

The ecosystem response to anthropogenic nitrogen (N) loading in estuarine systems is determined by hydrodynamics, biogeochemical transformation rates, and other system-specific characteristics. Historically, San Francisco (SF) Bay has been an outlier from other estuaries with an unusual resistance to eutrophication, despite having extremely high rates of nitrogen loading. Recent increases in phytoplankton biomass and an unprecedented harmful algal bloom, however, have increased the urgency to understand rates and drivers of nitrogen removal in the system. To assess benthic N cycling rates, we conducted seasonal measurements across nine sites in South and Lower South SF Bay, the two sub-embayments with the highest rates of area-normalized N loading, to determine the rates and potential drivers of denitrification and dissimilatory nitrate reduction to ammonium (DNRA). Denitrification rates averaged 60.6 ± 8.1 µmol m⁻² h⁻¹ and were primarily coupled to nitrification. Denitrification rates were positively correlated with DNRA rates and % clay. DNRA rates ranged from 0 to 20 µmol m⁻² h⁻¹ and on an annual basis averaged ~ 10% of total benthic nitrate reduction, with a negative correlation to % clay content in the surface sediment. The measured denitrification rates account for the removal of, on average, 14% of N loaded annually to South SF Bay, leaving a sizeable portion for alternate fates (e.g., recycling, export, or burial) and potential for substantial temporal and spatial variability (1–79%). This identifies the relative importance of sediment denitrification in ecosystems characterized by high nutrients and low productivity.

Subterranean estuaries (STEs) are critical ecosystems at the interface of meteoric groundwater and subsurface seawater that are threatened by sea level rise. To characterize the influence of tides and waves on the STE microbial community, we collected porewater samples from a high‐energy beach STE at Stinson Beach, California, USA, over the two‐week neap‐spring tidal transition during both a wet and dry season. The microbial community, analyzed by 16S rRNA gene (V4) amplicon sequencing, clustered according to consistent physicochemical features found within STEs. The porewater community harbored relatively abundant Proteobacteria, Verrucomicrobiota, and Bacteroidota, as well as members of the archaeal DPANN superphylum and bacterial Candidate Phyla Radiation (CPR). Tidal conditions were not associated with microbial community composition; however, a wave overtopping event significantly impacted the beach microbiome. As a baseline for environmental change, our results elucidate the unique dynamics of a STE microbiome with unprecedented temporal resolution, highlighting the transport of cellular material through beach porewater due to waves.

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle from ammonia-oxidizing Thaumarchaeota is currently considered the most energy-efficient aerobic carbon fixation pathway. The Nitrosopumilus maritimus 4-hydroxybutyryl-CoA synthetase (ADP-forming; Nmar_0206) represents one of several enzymes from this cycle that exhibit increased efficiency over crenarchaeal counterparts. This enzyme reduces energy requirements on the cell, reflecting thaumarchaeal success in adapting to low-nutrient environments. Here we show the structure of Nmar_0206 from Nitrosopumilus maritimus SCM1, which reveals a highly conserved interdomain linker loop between the CoA-binding and ATP-grasp domains. Phylogenetic analysis suggests the widespread prevalence of this loop and highlights both its underrepresentation within the PDB and structural importance within the (ATP-forming) acyl-CoA synthetase (ACD) superfamily. This linker is shown to have a possible influence on conserved interface interactions between domains, thereby influencing homodimer stability. These results provide a structural basis for the energy efficiency of this key enzyme in the modified 3HP/4HB cycle of Thaumarchaeota.

We use metagenome-assembled genomes (MAGs) to understand single-carbon (C1) compound-cycling—particularly methane-cycling—microorganisms in montane riparian floodplain sediments. We generated 1,233 MAGs (>50% completeness and 50% abundance in the MAG library) in one sample that also contained aceticlastic methanogens, indicating a potential C1/methane-cycling hotspot. Ca . Methylomirabilis MAGs from SR encode pathways for methylotrophy but do not harbor methane monooxygenase or nitrogen reduction genes. Comparative genomic analysis supports that one clade within the Ca . Methylomirabilis genus is not methanotrophic. The genetic potential for methylotrophy was widespread, with over 10% and 19% of SR MAGs encoding a methanol dehydrogenase or substrate-specific methyltransferase, respectively. MAGs from uncultured Thermoplasmata archaea in the Ca . Gimiplasmatales (UBA10834) contain pathways that may allow for anaerobic methylotrophic acetogenesis. Overall, MAGs from SR floodplain sediments reveal a potential for methane production and consumption in the system and a robust potential for methylotrophy. IMPORTANCE: The cycling of carbon by microorganisms in subsurface environments is of particular relevance in the face of global climate change. Riparian floodplain sediments contain high organic carbon that can be degraded into C1 compounds such as methane, methanol, and methylamines, the fate of which depends on the microbial metabolisms present as well as the hydrological conditions and availability of oxygen. In the present study, we generated over 1,000 MAGs from subsurface sediments from a montane river floodplain and recovered genomes for microorganisms that are capable of producing and consuming methane and other C1 compounds, highlighting a robust potential for C1 cycling in subsurface sediments both with and without oxygen. Archaea from the Ca . Methanoperedens genus were exceptionally abundant in one sample, indicating a potential C1/methane-cycling hotspot in the Slate River floodplain system.

Gemmatimonadota bacteria are widely distributed in nature, but their metabolic potential and ecological roles in marine environments is poorly understood. Here, we obtained 495 metagenome-assembled genomes (MAGs), and associated viruses, from coastal to deep-sea sediments around the world. We used this expanded genomic catalog to compare the protein composition, and update the phylogeny of these bacteria. The marine Gemmatimonadota are phylogenetically different from those previously reported from terrestrial environments. Functional analyses of these genomes revealed these marine genotypes are capable of degradation of complex organic carbon, denitrification, sulfate reduction, and oxidizing sulfide and sulfite. Interestingly, there is widespread genetic potential for secondary metabolite biosynthesis across Gemmatimonadota, which may represent an unexplored source of novel natural products. Lineages associated with coral reefs are enriched in genes encoding secondary metabolites, which are likely utilized for ecological interactions there. Furthermore, viruses associated with Gemmatimonadota have the potential to ‘hijack’ and manipulate host metabolism, including the assembly of the lipopolysaccharide in their hosts. This expanded genomic diversity advances our understanding of these globally distributed bacteria across a variety of ecosystems and reveals genetic distinctions between those in terrestrial and marine communities.

Transient or recurring blooms of ammonia-oxidizing archaea (AOA) have been reported in several estuarine and coastal environments, including recent observations of AOA blooms in South San Francisco Bay (SFB). Here, we measured nitrification rates, quantified AOA abundance, and analyzed both metagenomic and metatranscriptomic data to examine the dynamics and activity of nitrifying microorganisms over the course of an AOA bloom in South SFB during the autumn of 2018 and seasonally throughout 2019. Nitrification rates were correlated with AOA abundance in qPCR data and both increased several orders of magnitude between the autumn AOA bloom and spring and summer seasons. From bloom samples, we recovered an extremely abundant, high-quality Ca. Nitrosomarinus catalina-like AOA metagenome-assembled genome (MAG) that had high transcript abundance during the bloom and expressed >80% of genes in its genome. We also recovered a putative nitrite-oxidizing bacteria (NOB) MAG from within the Nitrospinaceae that was of much lower abundance and had lower transcript abundance than AOA. During the AOA bloom, we observed increased transcript abundance for nitrogen uptake and oxidative stress genes in non-nitrifier MAGs. This study confirms AOA are not only abundant, but also highly active during blooms oxidizing large amounts of ammonia to nitrite - a key intermediate in the microbial nitrogen cycle - and producing reactive compounds that may impact other members of the microbial community.